RIP-seq of translation initiation factors eIF2γ, eIF3b and two RBPs involved in the process of mRNA degradation and storage, Pat1p and Lsm1p
RIP-seq of translation initiation factors eIF2γ, eIF3b and two RBPs involved in the process of mRNA degradation and storage, Pat1p and Lsm1p
All yeast growth was in synthetic complete dextrose (SCD) media lacking histidine (ForMedium, UK) at 30 o C . BY4741 TAP tagged strains of Gcd11p, Lsm1p, Pat1p, and Prt1p were obtained from Thermo Scientific Open Biosystems (Waltham, MA, USA). Cells were grown to an OD 600 of 0.6 - 0.7, frozen in liquid nitrogen and lysed using a Spex freezer Mill 6870 into Lola140: 20 mM Tris - HCl (pH 8), 140 mM NaCl, 1 mM MgCl 2 , 0.5% NP40, 0.5 mM DTT, 1 mM PMSF, EDTA free Protease Inhibitor cocktail tablet (Roche Diagnostics, Indianaplois, IN, USA), 100 μM NaV 3 O 4 , 5 mM NaF and 40 units/ml RNasin (Promega, Flitchburg, WI, USA). RNA was isolated and purified using Trizol LS reagent (Invitrogen) from input yeast lysate or immunoprecipated TAP tagged protein, quantified using a N anodrop 8000 spectrophotometer (Thermo ) and rRNA depleted . rRNA depletion was confirmed using a Bioanalyzer (Agilent Technologies, CA, USA) Sequencing libraries were then generated using the TruSeq Stranded mRNA sample preparation kit ( Illumina , CA, USA). Total RNA was submitted to the University of Manchester Genomic Technologies Core Facility (GTCF). Quality and integrity of the RNA samples were assessed using a 2200 TapeStation (Agilent Technologies) and then libraries generated using the TruSeq® Stranded mRNA assay (Illumina, Inc.) according to the manufacturer's protocol. Briefly, rRNA depleted total RNA (0.1-4ug) was used as input material which was fragmented using divalent cations under elevated temperature and then reverse transcribed into first strand cDNA using random primers. Second strand cDNA was then synthesised using DNA Polymerase I and RNase H. Following a single 'A' base addition, adapters were ligated to the cDNA fragments, and the products then purified and enriched by PCR to create the final cDNA library.